The invention relates to the production of antibody formats and their immunological applications, more specifically in immunotherapy and immunodiagnostic. The inventors call to mind that antibody molecules are immunoglobulins (Ig) belonging to 5 classes: IgM, IgG, IgD, IgE and IgA. In general, these molecules comprise a heavy chain (H) and a light chain (L) that is either the kappa chain (κ), or the lambda chain (λ).
Each class of immunoglobulins comprises a specific type of H chain: μ chain for IgM, γ for IgG, δ for IgD, ε for IgE and α for IgA. Each chain is formed by domains, each with an inner disulphide bond. An L chain has two domains and an H chain has 4 domains. The sequence of the domain comprising the amine end of each chain is variable (VH and VL regions), that of other domains is constant (CH1, CH2 and CH3 of the H chain, and CL of the L chain). The variable regions V comprise regions of hypervariable sequences called CDR together determining the complementarity.
In the H chains, the first two domains (VH-CH1) are followed by a hinge region. In an immunoglobulin, the L chain is connected to the H chain by a disulphide bond to form a heterodimer. This heterodimer is connected to the same heterodimer by several disulphide bonds at the hinge region to form the immunoglobulin. By splitting with a protease at the level of the hinge, we obtain two fragments: fragment Fab (antigen binding domain, comprising the VL-CL and VH-CH1 domains) and fragment Fc (effector domain, comprising domains (CH2-CH3)2).
The invention more specifically refers to antibody fragments and different antibody formats created from these fragments, in particular formats of chimerised or humanised, multispecific and/or multivalent antibodies. The “antibody formats” as referred to in the invention correspond to different combinations of domains and regions of the types mentioned above.
By “chimerised antibody”, the inventors refer to a VH domain of animal origin fused to constant regions of human immunoglobulin. By “humanised antibody”, the inventors refer to a human VH domain on which hypervariable regions (CDRs) are grafted from a VH of animal origin, fused to constant regions of a human Ig.
These antibodies recognise the epitopes of targets corresponding to a given molecule. These epitopes may differ, and belong to different targets or the same target. Thereby “bispecific antibody” refers to a format with two different VH binding two different targets; “biepitopic antibody” refers to a format with two different VH binding two different epitopes on the same target. The “valence” corresponds to the number of times the same VH is found on the fragment considered.
The recognition specificity of antibodies to reach a determined target has been used for the diagnosis and treatment of different diseases and, in particular in oncology, where the target may be an antigen associated with a tumour, a growth factor receptor, an oncogene product or a muted “tumour suppressor” gene, or even a molecule linked to angiogenesis or a molecule also expressed on non-tumoural cells, but absent from progenitor cells (as in the case of CD20).
After over 20 years of experimental work, the immunotargeting of tumours by monoclonal antibodies is currently developing considerably. Thereby, the results of the different clinical studies have recently demonstrated the therapeutic possibilities of certain antibodies and have led to their approval by the FDA and the granting of a European AMM.
This progression is largely due to the use of so-called “second generation” recombinant antibodies: humanised antibodies, such as Herceptine, an anti-HER2/Neu antibody used in association with chemotherapy in certain breast carcinomas; and chimeric antibodies such as Rituximab, an anti-CD20 antibody used in the treatment of follicular B-cell lymphomas. Through gene engineering, it is possible to “graft” the variable or hypervariable regions of mouse antibodies on human Ig molecules. New techniques can now be used to obtain fully human antibodies either by selection of variable human domains expressed on phages (so-called “Phage display” technique), or by using transgenic mice producing human antibodies.
Moreover, the concept of bispecific antibodies has been used to stimulate the immune system and thereby favour the contact between the tumoural target cell and an effector cell. It consists in constructing an antibody endowed with a double specificity. This antibody should be able to bind a molecule produced at the surface of tumoural cells (such as CEA, HER2/Neu, GD2, etc.) and a molecule expressed at the surface of effector cells of the immunity, NK cells, killer T lymphocytes or CTL, polynuclear neutrophils, monocytes and macrophages (such as Fc receptors, etc.). A variant of this strategy consists of constructing an antibody linking a molecule produced at the surface of the tumoural cell and a molecule presenting direct or indirect properties of cytotoxicity (radio-element, toxin, prodrug).
Until now, most of the bispecific antibodies were developed by biochemically coupling 2 fragments of antibodies. However, this technique is rarely developed on an industrial scale. Several bispecific antibodies have been genetically developed, such as bispecific antibodies of the scFv type (“diabodies”). Unfortunately, they remain difficult to produce in E. coli in soluble form and they also are not very effective in terms of ADCC.
Within the search for candidate antibodies to generate antibody formats for immunotherapy and in particular to obtain multi-specific antibodies, the inventors directed their work towards specific antibodies, without a light chain, identified in the Camelidae (camel, dromedary, llama) (Hamers-Casterman et al., 1993).
Variable domains of heavy single chain antibodies from Camelidae (VHH), specifically recognising a type of antigen, were selected from immunised animals and were used to develop different formats of chimerised or humanised antibodies that may be produced from plasmid constructions. It turned out that the different formats were compatible to enable the production of any other VHH or humanised VHH, or human VH.
The invention aims at providing antibody formats comprising a part of the totality of VHH or humanised VHH, or human VH domains with properties to recognise the searched for targets and epitopes. It also aims at providing a method for the production of these different constructions. According to another aspect, the invention aims at immunotherapeutic and immunodiagnostic applications of the different formats provided. The invention also relates to antibody formats including a part or the totality of the VHH domains of Camelidae, in particular llamas and/or human VH, fused to constant regions of human antibodies.
According to a first means of achievement of the invention, the antibody formats are of Fab type and are characterised by the association of two identical or different VHH domains or two human VH domains, or two human VH domains on which are grafted the CDRs of the VHH, one of the domains being fused to the constant region Cκ or Cλ of a human immunoglobulin, the other to the constant region CH1 from a human immunoglobulin.
According to a second means of achievement of the invention, the antibody formats are of the Fab′ type and are characterised by the association of two identical or different VHH domains or two human VH domains, or two human VH domains on which are grafted the CDRs of the VHH, one of the domains being fused to the constant region Cκ or Cλ of a human immunoglobulin, the other to the constant region CH1 followed by a hinge region H from a human immunoglobulin. These chimerised or humanised antibody formats are of monospecific/bivalent, bispecific/monovalent and biepitopic/monovalent types.
According to a third means of achievement of the invention, the antibody formats are of F(ab′)2 type and are characterised by the association of two formats of Fab′ type as defined above. These chimerised or humanised antibody formats have a hinge region H, from a human immunoglobulin and allow for monospecific/tetravalent, bispecific/bivalent and biepitopic/bivalent combinations.
According to a fourth means of achievement of the invention, the antibody formats are of F(ab′)2 type and are characterised by the association of two Fab′ obtained by reduction of formats of the above F(ab′)2 type. These chimerised or humanised antibody formats have a hinge region H from a human immunoglobulin and allow for monospecific/tetravalent to tetraspecifique/monovalent or tetraepitopic/monovalent combinations, including all of the intermediate possibilities.
According to a fifth means of achievement of the invention, the antibody formats are of (HCH2CH3)2 type (H, representing the hinge region of a human immunoglobulin, CH2 and CH3, representing the second and third constant domain of a heavy chain from a human Ig, and are characterised by the association of two identical VHH or human VH, or two human VH on which are grafted the hypervariable regions of the VHH, each being fused at the region H—CH2-CH3 of a human Ig. These chimerised or humanised antibody formats allow for monospecific/bivalent combinations.
According to a sixth means of achievement of the invention, the antibody formats are of mAb* type (this type refers to variable domains of origin replaced by all or part of the VHH or humanised VHH or human VH domains, fused to constant regions of human antibodies) and are characterised by the association of two identical or different VHH or two human VH, or two human VH on which are grafter hypervariable regions of VHH, one being fused to the Cκ or human Cλ region, the other to the CH1-H—CH2-CH3 region of a human Ig. These chimerised or humanised antibody formats allow for monospecific/tetravalent and bispecific/bivalent and biepitopic/bivalent combinations.
In these different formats, the immunoglobulin is an IgG, corresponding to a human isoform IgG1, IgG2, IgG3 or IgG4, or a human IgA corresponding to an isoform IgA1, IgA2, or any other human Ig. The VHH may be replaced by human VH or humanised VHH by the grafting of CDRs from VHH on human VH.
In the examples of the above means of achievement of the invention, the VHH correspond to or comprise fragments of Camelidae VHH antibodies, in particular from llamas. In particular, it involves characteristic fragments in that it consists of a part or the totality of anti-carcinoembryonic antigen (anti-CEA in abbreviated form) or anti-receptor FcγRIII (anti-CD16 in abbreviated form) fragments.
The anti-CEA antibody fragments more specifically comprise an amino acid sequence selected from the group consisting of the sequences SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:105. The anti-CD16 antibody fragments in a preferred manner comprise an amino acid sequence selected from the group consisting of the sequences SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:103 and SEQ ID NO:104. These fragments form new products and in this way also come into the scope of the invention.
The invention also includes the CDRs of these VHH fragments. The invention also includes a method for the production of chimerised or humanised, multispecific and/or multivalent antibodies for immunotherapy or immunodiagnostics, characterised in that it comprises the use of antibody formats defined above. The invention specifically aims at a method of said formats comprising anti-CEA and anti-CD16 Camelidae VHH, in particular llama VHH. More specifically, it refers to variable domains of anti-CEA and anti-CD16 VHH advantageously produced according to a protocol comprising: the immunisation of Camelidae, in particular of llamas with, as immunogen, a CEA or a CD 16; the purification of B lymphocytes obtained from blood; the construction of a VHH bank; and the isolation of VHH from the bank.
The construction of the bank comprises: the extraction of whole RNA from B lymphocytes; the reverse transcription of RNA to obtain the corresponding cDNA; the amplification by PCR of genes coding for the variable regions of single heavy chain anti-CD 16 and anti-CEA antibodies; and the ligation of VHH DNA fragments obtained by cutting, by enzymes, of DNA amplified with a phagemid. The VHH are isolated from banks by the phage display technique and are purified.
Said variable domains of anti-CEA and anti-CD16 VHH are advantageously produced according to a protocol comprising: the immunisation of Camelidae, in particular llamas with, as immunogen, a CEA or a CD16; the purification of B lymphocytes recovered from blood; the construction of a VHH bank; and the isolation of VHH from the bank.
In an advantageous manner, the construction of the bank comprises: the extraction of whole RNA from B lymphocytes; the reverse transcription of RNA to obtain the corresponding cDNA; the amplification by PCR of genes coding for the variable regions of single heavy chain anti-CD16 and anti-CEA antibodies; and the ligation of fragments of DNA VHH, obtained by cutting by enzymes of amplified DNA with a phagemid. The VHH are isolated from banks by the phage display technique and are purified. The different VHH have been validated in terms of specificity and affinity as illustrated by the examples.
According to the invention, the genes of the selected VHH are then introduced in expression vectors, in particular plasmids, to produce different chimerised multispecific and/or multivalent (anti-CEA/anti-CD16) antibodies, able to bind with tumoral cells expressing the CEA at their surface and recruit the effector cells from the immune system (monocytes, macrophages, NK, polynuclear neutrophils, et al.) that express CD16.
The invention also refers to expression vectors of the antibody formats defined above. It more specifically refers to expression vectors, in particular plasmids containing, between two unique sites of restriction enzymes, the promoters, the signal sequences, the nucleotide sequences able to code for the VHH domains defined above, and the constant regions of a human Ig, or for human VH domains, the CDRs regions of a VHH, and the constant regions of a human Ig.
The plasmids according to the invention are able to express high quantities of the antibody formats defined above, in soluble forms in bacteria and the regions coding for the antibody domains may easily be transferred to other systems of prokaryotic or even eukaryotic expression.
The invention therefore refers to plasmids pCκCH1γ1-TAG (SEQ ID NO:98 and SEQ ID NO:112) and pCκCH1γ1 (SEQ ID NO:100 and SEQ ID NO:114) allowing for the production of antibodies of Fab type according to a first means of achievement of the antibody formats defined above. These plasmids are more specifically characterised by the insertion of nucleotide sequences coding for the light region Cκ, and the constant heavy region CH1 of an Ig in the plasmid p55Flag/RBS/35cmyc6HisGS (SEQ ID NO:94 and SEQ ID NO:110).
The invention also refers to the plasmids pCκCH1 Hγ1-TAG (SEQ ID NO:99 and SEQ ID NO:113) and pCκCH1Hγ1 (SEQ ID NO:101 and SEQ ID NO:115) allowing for the production of antibodies of Fab′ and F(ab′)2 type according to a second, third and fourth means of achievement of the antibody formats defined above. These plasmids are more specifically characterised by the insertion of nucleotide sequences coding for the heavy chain CH1 and the hinge region (H) of an Ig in p55CκFlag/RBS/35cmyc6HisGS (SEQ ID NO:97 and SEQ ID NO:111).
The invention also refers to the plasmids pHCH2CH3γ1-TAG (SEQ ID NO:95) and pHCH2CH3γ1 (SEQ ID NO:96) allowing for the production of antibodies of (HCH2CH3)2 type according to a fifth means of achievement of the antibody formats defined above. These plasmids are more specifically characterised by the insertion of nucleotide sequences coding for the hinge region (H) and the constant regions CH2 and CH3 of an Ig in p55Flag/RBS/35cmyc6HisGS.
The invention also refers to plasmid pMabγI* (SEQ ID NO:102 and SEQ ID NO:116) allowing for the production of antibodies of mAb* type according to a sixth means of achievement of the invention. This plasmid is more specifically characterised by the insertion of nucleotide sequences coding for the constant heavy region CH1, the hinge region and the constant regions CH2 and CH3 of an Ig in pCκCH1γ1-TAG.
The diagrams of these plasmids are illustrated in FIG. 10B and their nucleotide sequences in FIG. 11. The intermediate plasmids used for the construction of the above plasmids also fall within the scope of the invention. More specifically, it involves plasmids p55PhoA6HisGS/N− (SEQ ID NO:89), p55PhoA6HisGS/NAB′ (SEQ ID NO:90), p55/MCS1 (SEQ ID NO:92), p55Flag/RBS/35 (SEQ ID NO:93 and SEQ ID NO:109), p55Flag/RBS/35cmyc6HisGS (SEQ ID NO:94 and SEQ ID NO:110) and p55CκFlag/RBS/35cmyc6HisGS (SEQ ID NO:97 and SEQ ID NO:111) constructed to develop the plasmids defined above. The domains CH1, CH2, CH3, H of an Ig in these plasmids belong to IgGI, IgG2, IgG3 or IgG4, or even IgA, or any other Ig.
The genes coding for the VHH or the human VH are introduced between the unique sites in the different plasmids. These genes may be replaced by genes coding for humanised VHH by grafting of CDRs of VHH on human VH. More generally, the plasmids used according to the invention may be designed to contain nucleotide sequences coding for VHH other than anti-CEA or anti-CD16 VHH, or for other human VH, or for other humanised VHH, able to bind on any molecule.
The invention also refers to plasmid p55PhoA6HisGS−/NAB− (SEQ ID NO:91) characterised in that it comprises the nucleotide sequences to produce human VH domains fused to alkaline phosphatase according to the diagram in FIGS. 10A and 11.
A method to select the variable human fragments of heavy chains of immunoglobulins (VH) and isolate the best produced and best secreted clones has been developed. Advantageously, these human VH are used as a matrix to graft the CDR from previously selected VHH in order to humanise the variable regions.
The antibody formats defined above are of great interest in immunotherapy and immunodiagnostics. They are able to recognise different molecules or bind two different epitopes on the same molecule, and also provide access to new epitopes that are not recognised by the concentional antibodies. They may also be humanised, which opens the way to advantageous prospects, to have antibodies of low immunogenicity after injection in man. The fact that they are obtained in a soluble form is an additional characteristic of interest for these antibodies. Their applications in immunodiagnostics and immunotherapy are also part of the invention.